When a pyrimidine residue in cellular DNA becomes modified by oxidation, reduction or hydration of its 5,6 double bond, repair is initiated by a DNA-glycosylase activity which cleaves the N-glycosyl bond of the damaged residue releasing the modified base and creating an abasic (AP) site in the DNA backbone. Such DNA glycosylase activities have been identified in bacteria, yeast and mammalian species Brent, Biophys. J., 13:399-401 (1973); Bacchetti, et al., Biochim. Biophys. Acta, 390:285-297 (1975); Duker, et al., Nature, 255:82-84 (1975); Ness. et al., Biochim. Biophys Acta, 520:111-121 (1978); Demple, et al., Nature, 287:203-208 (1980): Cunniningham, et al., Proc. Natl. Acad. Sci. U.S.A., 82:474-478 (1985); Doetsch. et al., Biochemistry, 25:2212-2220 (1986); Boorstein, et al., Biochemistry, 28:6164-6170 (1989)!. The DNA repair enzyme E. coli endonuclease III was the first of such enzymes to be described. It was identified not on the basis of its DNA glycosylase activity, but rather by its nicking activity directed against UV-irradiated DNA Radman, J. Biol. Chem., 251:1438-1445 (1976)!. Subsequently, it was shown that nicking of UV-irradiated DNA resulted from 2 enzymatic activities; a DNA-glycosylase which released pyrimidine (cytosine and/or uracil) hydrates from the DNA backbone, yielding an apyrimidinic (AP) site Boorstein, et al., 1989, supra!, and an activity which effected strand cleavage via .beta.-elimination of the 3' phosphate group of the apyrimidinic sugar residue Bailly, et al., Biochemical J. 242:565-572 (1987); Kim, et al., J. Biol. Chem., 264:2739-2745 (1989); Mazumder, et al., Biochemistry, 30:1119-1126 (1991)!. The latter activity has been termed an AP lyase to distinguish it from AP endonucleases, such as exonuclease III or endonuclease IV, which catalyze strand cleavage via hydrolysis of phosphodiester bonds Bailly, et al., Nucleic Acids Res., 17:3617-3618 (1989)!. Endonuclease III is one of a group of enzymes, including T4 endonuclease V and the E. coli Fpg protein (MutM), which demonstrate both DNA-glycosylase and AP lyase activities Demple, et al., Ann. Rev. Biochem., 63:915-948 (1994); Dodson, et al., J. Biol. Chem., 269:32709-32712 (1994)!.
In addition to excising pyrimidine hydrates, the DNA-glycosylase activity of endonuclease III also excises pyrimidine glycols, ring-contracted pyrimidine derivatives, such as 5-hydroxymethylhydantoin, and urea residues composed of the N1-C2-N3 atoms of the pyrimidine skeleton Strniste, et al., Proc. Natl. Acad. Sci. U.S. A., 72:1997-2001 (1975); Demple, et al., 1980, supra; Breimer, et al., J. Biol. Chem., 259:5543-4458 (1984); Cunningham, et al., 1985, supra!. Enzyme activities functionally analogous to endonuclease III have been identified in bacteria other than E. coli, in yeast, and in mammalian cells and tissues through the use of UV-irradiated, chemically oxidized, and .gamma.-irradiated DNA as substrates Brent, 1973 supra; Bacchetti, et al., 1975, supra; Duker, et al., 1975, supra; Ness, et al., 1978, supra, Doetsch, et al., 1986, supra!. Extracts of Hela cells have been shown to contain a thymine glycol DNA-glycosylase Higgins, et al., Biochemistry, 26:1683-1688 (1987)!. It has also been demonstrated that both endonuclease III and Hela cell extracts released cytosine hydrate (as well as its deamination product, uracil hydrate) from UV-irradiated DNA Boorstein, et al., 1989, supra!. Kim, et al. 1989, supra! described 2, or possibly 3, UV-endonuclease activities in Hela cells by monitoring the nicking of UV-irradiated circular DNA. Huq et al. Eur. J. Biochem, 206:833-839 (1992)! reported a 25 fold purification of an endonuclease III-like activity from calf thymus and stated that the N-terminal sequence of this protein was not homologous to other known proteins.
In view of the significance and activity of endonuclease III as recited in the literature, it would be desirable, and a need therefore exists, to elucidate the mammalian homologs, and other potentially active fragments, that may be applied to the development of both diagnostic and therapeutic modalities, to treat the adverse effects of exposure to radiation and oxidation. It is therefore toward the fulfillment of this need that the present invention is directed.